3Departments of Neurology, 5Neurological Surgery, and 7The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, California 94143

Abstract

Excitatory and inhibitory balance of neuronal network activity is essential for normal brain function and may be of particular importance to memory. Apolipoprotein (apo) E4 and amyloid-β (Aβ) peptides, two major players in Alzheimer's disease (AD), cause inhibitory interneuron impairments and aberrant neuronal activity in the hippocampal dentate gyrus in AD-related mouse models and humans, leading to learning and memory deficits. To determine whether replacing the lost or impaired interneurons rescues neuronal signaling and behavioral deficits, we transplanted embryonic interneuron progenitors into the hippocampal hilus of aged apoE4 knock-in mice without or with Aβ accumulation. In both conditions, the transplanted cells developed into mature interneurons, functionally integrated into the hippocampal circuitry, and restored normal learning and memory. Thus, restricted hilar transplantation of inhibitory interneurons restores normal cognitive function in two widely used AD-related mouse models, highlighting the importance of interneuron impairments in AD pathogenesis and the potential of cell replacement therapy for AD. More broadly, it demonstrates that excitatory and inhibitory balance are crucial for learning and memory, and suggests an avenue for investigating the processes of learning and memory and their alterations in healthy aging and diseases.

Behavioral tests.

Behavioral tests were performed for MGE cell-transplanted and control-transplanted mice at 70–80 d after transplantation (DAT). All mice were singly housed during behavioral tests. The Morris water maze (MWM) test was conducted in a pool (122 cm in diameter) with room temperature water (22–23°C) with a 10 cm2 platform submerged 1.5 cm below the surface of opaque water during hidden trials (Andrews-Zwilling et al., 2010; Leung et al., 2012). Mice were trained to locate the hidden platform over four trials per day on hidden platform days 1–5 (HD1–5), where HD0 was the first trial on the first day, with a maximum of 60 s per trial. Each memory trial was conducted for 60 s in the absence of the platform at 24, 72, and 120 h after the final learning session. Memory was assessed as the percentage of time spent in the target quadrant that contained the platform during the learning trials compared with the average percentage of time spent in the nontarget quadrants. For visible trials, a black and white-striped mast (15 cm high) marked the platform location. The platform location and room arrangement remained constant throughout the assay with the exception of moving the platform during the visible trials. Speed was calculated by distance traveled divided by trial duration. Performance was objectively monitored using EthoVision video-tracking software (Noldus Information Technology). The open field test assesses habituation and general activity behavior by allowing the mice to explore a new, but empty, environment (Andrews-Zwilling et al., 2012). After at least 2 h of room habituation, mice were placed in an odor-standardized chamber cleaned with 30% EtOH for 15 min. Activity behavior was monitored and analyzed by software from San Diego Instruments. The elevated plus maze evaluates anxiety and exploratory behavior by allowing mice to explore an open, illuminated area (open arm) or hide in a dark, enclosed space (closed arm; Bien-Ly et al., 2011). Here, mice were placed in an odor-standardized maze cleaned with 30% EtOH for 10 min after at least 2 h of room habituation. Behavior was analyzed by infrared photo cells interfacing with Motor Monitor software (Kinder Scientific).

Spontaneous EPSCs and IPSCs were recorded in the same cells by voltage-clamping the membrane potential at the reversal potential of the GABAergic current (−50 mV) and glutamatergic current (10 mV), respectively. To stabilize recordings at the depolarizing membrane potential, patch pipettes were filled with an internal solution containing the following: 120 mm CsMeSO3, 0.5 mm EGTA, 10 mm BAPTA, 10 mm HEPES, 2 mm Mg-ATP, 0.3 mm Na-GTP, and 5 mm QX-314. Events were sampled for 200 s and detected with a cutoff of ±5 pA.

Intrinsic excitability of interneurons was assessed by measuring the firing rate in response to a series of depolarizing current injections (1 s; 0.05–1 nA). Patch pipettes were filled with an internal solution containing the following: 100 mm K-gluconate, 20 mm KCl, 10 mm HEPES, 4 mm Mg-ATP, 0.3 mm Na-GTP, 10 mm phosphocreatine, and 0.2% biocytin. After the experiment, slices were fixed and stained for biocytin, somatostatin, and parvalbumin to confirm the identity of the recorded cells.

Image collection and cell quantification.

Histological images were collected using a Biorevo BZ-9000 Keyence digital microscope, a Leica spinning disk confocal microscope, or a Bio-Rad scanning confocal microscope. Quantification of interneuron subtypes was obtained manually by observations performed on an upright epifluorescent DM500B Leica microscope and on a Keyence digital microscope. Quantification of microglia (markers of Iba1 and CD68) was obtained using the semiautomated Image-based Tool for Counting Nuclei (ITCN) plugin in ImageJ for images covering 0.5 mm2 of the dentate gyrus. Quantification of GAD67+ and GFP+ cells was conducted on every 10th section (30 μm) across the whole hippocampus using the semiautomated ITCN plugin in ImageJ for captured images.

Statistical analysis.

All statistical analyses for electrophysiology were performed using IGOR Pro software (WaveMetrics), and all other analyses were performed using Prism 6 software (GraphPad). Differences between means were assessed by t test, one-way ANOVA, or repeated-measures ANOVA, followed by Bonferroni or Tukey-Kramer post hoc tests, as noted in text and figure legends. In all cases, a p value of <0.05 was considered to be statistically significant. All error bars represent ±SEM.

Immunofluorescent analyses of neuronal subtypes derived from transplanted MGE cells in the hippocampal hilus of apoE4-KI mice at 80–90 DAT. A, Transplanted MGE cells were stained positive for NeuN and GABA. B, Immunofluorescent costaining of inhibitory interneuron subtypes positive for SOM, NPY, and PV. C–E, Immunostaining of cells that were positive for ChAT (C), GFAP (D), or Olig2 (E). F, Quantification of the percentage of GFP+ cells that were also positive for different cell markers (n = 5–8 sections per brain, 3–5 mice per group for each cell marker). Values are shown as the mean ± SEM. Scale bar, 50 μm.

Immunofluorescent analyses of neuronal subtypes derived from transplanted MGE cells in the hippocampal hilus of apoE3-KI mice at 80–90 DAT. A, Transplanted MGE cells were stained positive for NeuN and GABA. B, Immunofluorescent costaining of inhibitory interneuron subtypes positive for SOM, NPY, and PV. C–E, Immunostaining of cells that were positive for ChAT (C), GFAP (D), or Olig2 (E). F, Quantification of the percentage of GFP+ cells that were also positive for different cell markers (n = 5–8 sections per brain, 3–5 mice per group for each cell marker). Values are shown as the mean ± SEM. Scale bar, 50 μm.

Dysfunction of the GABAergic system may also contribute to cognitive impairment in humans. AD patients have decreased GABA and somatostatin levels in the brain and CSF (Davies et al., 1980; Bareggi et al., 1982; Zimmer et al., 1984; Hardy et al., 1987; Seidl et al., 2001), and these alterations were more severe in apoE4 carriers (Grouselle et al., 1998). ApoE4 is associated with increased brain activity at rest and in response to memory tasks (Filippini et al., 2009; Dennis et al., 2010), possibly reflecting impaired GABAergic inhibitory control. Furthermore, GABA levels in human CSF decrease with age (Bareggi et al., 1982)—the strongest risk factor for AD. Thus, aging- and AD-related memory deficits in humans may also result from an excitatory–inhibitory imbalance of the hippocampal dentate gyrus due to inhibitory interneuron dysfunction or loss (Palop and Mucke, 2010; Huang and Mucke, 2012). The current study provides a proof of concept for developing inhibitory interneuron replacement therapies for treatment of AD. In support of this possibility, a recent study (Liu et al., 2013) showed that transplantation of human embryonic stem cell-derived MGE-like cells rescued learning and memory deficits induced by acute hippocampal lesions in mice.

Strikingly, the grafted MGE-derived inhibitory interneurons not only survive and functionally integrate in the hippocampus of aged mice (12–17 months of age) but also do so in an apparently toxic environment—the presence of apoE4 and Aβ accumulation. Since wild-type mouse MGE-derived GABAergic interneuron progenitors, which express wild-type mouse apoE, survive and integrate equally well in the hippocampal hilus of apoE3-KI and apoE4-KI mice for >3 months, this suggests that the detrimental effect of apoE4 on hilar GABAergic interneurons is cell autonomous. The observation that the wild-type mouse MGE cells also survive and integrate well in the hippocampal hilus of apoE4-KI mice with significant Aβ plaque buildup further supports this conclusion. This is important for potential stem cell-based therapy of AD in the future, indicating that transplanted human MGE-like cells without apoE4 expression or Aβ overproduction would have a good chance to survive and functionally integrate in the brains of AD patients.

Interestingly and importantly, the transplanted MGE cells appear to have a minimal effect on learning and memory in apoE3-KI recipients with normal cognition. We previously reported a threshold number of hilar GABAergic interneurons (∼2500–3000) that appeared to distinguish normal versus impaired learning and memory in female apoE4-KI mice at 16 months of age (Andrews-Zwilling et al., 2010; Leung et al., 2012). At this age, all female apoE3-KI mice had >3000 hilar GABAergic interneurons (with a wide range of 3000–5200 interneurons), whereas some of the female apoE4-KI mice had <3000 interneurons and had greater learning deficits. Thus, adding ∼1000 hilar interneurons by MGE cell transplantation significantly improved learning and memory performance in female apoE4-KI mice. Since there was no correlation between hilar GABAergic interneuron numbers and normal learning and memory performance in apoE3-KI mice with a wide range of hilar interneurons (3000–5200; Andrews-Zwilling et al., 2010; Leung et al., 2012), it is not surprising that hilar MGE cell transplantation, which adds ∼1000 interneurons, has a minimal effect on normal learning and memory in apoE3-KI mice.

To date, all efforts to develop therapies that target specific AD-related pathways have failed in human trials of late-stage AD (Huang and Mucke, 2012). As a result, an emerging consensus in the field is that treatment of moderate-to-advanced AD patients with current drugs comes too late, probably due to neuronal loss in the hippocampus (Huang and Mucke, 2012). In this regard, cell replacement therapies, such as using human embryonic stem cell-derived or induced pluripotent stem cell-derived MGE-like cells (Liu et al., 2013), hold a potential for the treatment of patients with moderate-to-advanced AD. A key aspect of hilar GABAergic interneurons is that one such inhibitory neuron connects to and thus influences >1500 excitatory granule neurons in the dentate gyrus (Morgan et al., 2007). This suggests that even if a small number of the transplanted therapeutic cells survive and functionally integrate, they could make a significant functional improvement in the dentate gyrus and thus in learning and memory. Our findings that ∼1000 functionally integrated transplanted GABAergic interneurons are sufficient to rescue apoE4- and Aβ-induced learning and memory deficits in two widely used AD-related mouse models strongly support this notion.

Footnotes

This work was supported in part by Grant RN2-00952 from the California Institute for Regenerative Medicine, Grant AG022074 from the National Institutes of Health, the S.D. Bechtel, Jr. Foundation, the Roddenberry Foundation, and the Hellman Foundation. We thank Laura Leung, Gui-Qiu Yu, Mercedes Paredes, and Derek Southwell for technical advice; Ravikumar Ponnusamy and Iris Lo for assistance on behavioral tests; Diane Nathaniel for electrophysiological data analysis support; Teodoro Meneses for animal husbandry; and Gary Howard, Laura Leung, and Philip Nova for editorial assistance.

Correspondence should be addressed to Dr. Yadong Huang,
Gladstone Institute of Neurological Disease, University of California, San Francisco, San Francisco, CA 94158.yhuang{at}gladstone.ucsf.edu